Accepted Manuscript

Short communication

Molecular phylogeny and diversification of the genus Odorrana (Amphibia,

Anura, Ranidae) inferred from two mitochondrial genes

Xiaohong Chen, Zhuo Chen, Jianping Jiang, Liang Qiao, Youqiang Lu, Kaiya

Zhou, Guangmei Zheng, Xiaofei Zhai, Jianxin Liu

PII: S1055-7903(13)00300-X

DOI: http://dx.doi.org/10.1016/j.ympev.2013.07.023

Reference: YMPEV 4673

To appear in: Molecular Phylogenetics and Evolution

Received Date: 19 March 2013

Revised Date: 14 July 2013

Accepted Date: 22 July 2013

Please cite this article as: Chen, X., Chen, Z., Jiang, J., Qiao, L., Lu, Y., Zhou, K., Zheng, G., Zhai, X., Liu, J.,

Molecular phylogeny and diversification of the genus Odorrana (Amphibia, Anura, Ranidae) inferred from two

mitochondrial genes, Molecular Phylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/j.ympev.

2013.07.023

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1

Molecular phylogeny and diversification of the genus Odorrana

(Amphibia, Anura, Ranidae) inferred from two mitochondrial genes

Xiaohong Chena,*, Zhuo Chen

a, Jianping Jiang

b, Liang Qiao

a, Youqiang Lu

a, Kaiya Zhou

c,*,

Guangmei Zhengd,

*, Xiaofei Zhaia, Jianxin Liu

e

Abstract

A diversity of hypotheses have been proposed for phylogenetic relationships and taxonomy

within the genus Odorrana, and great progress has been made over the past several decades.

However, there is still some controversy concerning relationships among Odorrana species.

Here, we used many paratypes and topotypes and utilized 1.81 kb of mitochondrial sequence

data to generate a phylogeny for approximately 4/5 of Odorrana species, and Odorrana

haplotypes form a strongly supported monophyletic group relative to the other genera

sampled. The deepest phylogenetic divergences within Odorrana separate three lineages

whose interrelationships are not recovered with strong support. These lineages include the

ancestral lineage of O. chapaensis, the ancestral lineage of a strongly supported clade

comprising many western species, and the ancestral lineage of a strongly supported clade

comprising all other Odorrana sampled. Within the latter clade, the first phylogenetic split

separates O. ishikawae from a well-supported clade comprising its other species. These

divergences likely occurred in the middle Miocene, approximately 12-15 million years ago.

Separation of the ancestral lineage of Odorrana from its closest relative, Babina in our study,

likely occurred in the early Miocene or possibly late Oligocene. Rates of lineage

accumulation remained high from the middle Miocene through the Pleistocene.

a College of Life Sciences, Henan Normal University, Xinxiang 453007, China

b Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

c Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences,

Nanjing Normal University, Nanjing 210046, China

d College of Life Sciences, Beijing Normal University, Beijing 100875, China

e Wuchuan High school, Wuchuan 564300, China

2

* Corresponding authors:

Xiaohong Chen

Tel: +86-373-3326340

Fax: +86-373-3329102

E-mail: xhchen-xx@sohu.com

Kaiya Zhou

Tel: +86-25-83598147

Fax: +86-25-85891526

E-mail: kyzhounj@126.com

Guangmei Zheng

Tel/ Fax: +86-10-58808988

E-mail: zhenggm@bnu.edu.cn

Keywords: Ranidae; Odorrana; Phylogeny; Diversification; Divergence date

1. Introduction

The genus Odorrana (Fei et al., 1990), consisting of approximately 53 species, is

endemic to East and Southeast Asia (Frost, 2013). The taxonomy and phylogeny of Odorrana

have long attracted the interest of evolutionary biologists and have been investigated using

both morphological and molecular data (Dubois, 1992; Fei et al., 1990, 2009, 2010; Ye and

Fei, 2001; Bain et al., 2003; Chen et al., 2005; Jiang and Zhou, 2005; Frost et al., 2006;

Matsui et al., 2005; Cai et al., 2007; Che et al., 2007; Stuart, 2008; Wiens et al., 2009;

Kurabayashi et al., 2010; Pyron and Wiens, 2011). Although considerable progress has been

made in the past two decades toward advancing our understanding of Odorrana

phylogenetics and taxonomy, these studies have left unresolved issues and also created some

new controversies. One controversy, for example, was the status of Odorrana. This genus

was first recognized by Fei et al. (1990) with the type species Odorrana margaretae, while

Dubois (1992) treated Odorrana as a subgenus of the genus Rana and erected a new

3

subgenus R. (Eburana) that included R. (E.) ishikawae, R. (E.) ijimae, R. (E.) narina, R. (E.)

swinhoana and R. (E.) livida with the type species R. (E.) narina. Later, Frost et al. (2006)

greatly expanded the genus Huia to include both Odorrana and R. (Eburana) based only on

the analysis of four species: H. nasica, Amolops chapaensis, R. (E.) chloronota and O.

grahami. However, subsequent analyses of mtDNA data (Jiang and Zhou, 2005; Matsui et al.,

2005; Cai et al., 2007), nuclear data (Stuart, 2008) and combinations of mtDNA and nuclear

data (Che et al., 2007; Wiens et al., 2009; Pyron and Wiens, 2011) supported monophyly of

Odorrana and rejected R. (Eburana) and Huia. Additionally, the systematic status and

phylogenetic position of some taxa such as O. ishikawae and O. chapaensis remain

controversial (Fig. 1), despite a variety of studies using a diverse array of systematic markers

(Matsui et al., 2005; Cai et al., 2007; Stuart, 2008; Wiens et al., 2009), even in complete

mitogenomes (Kurabayashi et al., 2010) and large concatenations of data (Pyron and Wiens,

2011). Additional conflicts regarding the relationships and taxonomies exist within the genus

Odorrana, where previous phylogenetic hypotheses contradicted one another (Fig. 1).

In this study, we sampled approximately 4/5 of the named species and two unnamed

species with many paratypes and topotypes for Odorrana based on the analysis of 1.81 kb of

molecular sequence data from two mitochondrial genes (12S and 16S rRNA) to present a

molecular phylogeny of Odorrana. The resulting phylogeny was used to test the validity of

Odorrana based on extensive taxonomic sampling. In addition, Bayesian relaxed-clock

estimation was performed to improve our understanding of the Odorrana radiation.

2. Materials and Methods

2.1. Taxon sampling, DNA extraction, amplification, and sequencing

Tissue samples from a total of 38 species, including 30 Odorrana specimens and 8

outgroup species, were collected for DNA sequencing. Additional sequence data of 13

Odorrana species and two other ranids were obtained from GenBank (Table 1). Specimen

data (species names, sampling localities, specimen voucher no. and GenBank Accession

Numbers) are given in Table 1, and geological distributions of all sampling sites are

presented in Fig. 2.

Total genomic DNA was extracted from thigh muscle or liver using a DNeasy Tissue

4

Extraction Kit (Qiagen) or with a standard phenol/chloroform procedure followed by ethanol

precipitation (Sambrook and Russell, 2006). Two fragments of 12S and 16S rRNA genes

were amplified using Ex-Taq DNA polymerase (TaKaRa) under the following conditions: 35

cycles at 95C for 5 min, 95C for 40 s, 47C -57C for 40-50 s, and 72C for 45-90 s

followed by a 10-min extension at 72C. Primer information is given in Table S1 in Appendix

A. The amplified PCR products were purified and sequenced in both directions with an ABI

3730 automated genetic analyzer. Novel sequences were deposited in GenBank under

accession numbers KF184996-KF185067 (Table 1).

2.2. Sequence alignment and phylogenetic analyses

Sequences from the 12S and 16S rRNA genes were separately aligned in Clustal X 1.81

(Thompson et al., 1997) with default parameters, and the software GBlocks (Castresana,

2000) was used under default settings to delete regions of ambiguous alignment (the

alignment file is available in TreeBase http://purl.org/phylo/treebase/phylows/study/TB2:

S14355). Further saturation testing was performed using DAMBE (Xia, 2003).

Phylogenies were built using maximum likelihood algorithms in MetaPIGA 2.0 (Helaers

and Milinkovitch, 2010) and Bayesian inference (BI) in MrBayes 3.1.2 (Huelsenbeck and

Ronquist 2001) for each gene independently and for a combined dataset of 12S and 16S

rRNA. Modeltest 3.7 (Posada and Crandall, 1998) was used to select the optimal models for

each partition based on the Akaike Information Criterion (AIC). Maximum Likelihood

analyses were performed using MetaPIGA 2.0 with 1000 replicate metaGA searches. The

Bayesian analyses of the nucleotide matrix were performed using the GTR + I + G model.

Four Markov chains were run for 20 million generations with sampling every 1000

generations. The stationarity of the likelihood scores of sampled trees was determined in

Tracer 1.4 (Rambaut and Drummond 2007). The first 10% of trees were removed as the

“burn-in” stage followed by calculation of Bayesian posterior probabilities (PP) and the 50%

majority-rule consensus of the post burn-in trees sampled at stationarity. Phylogenetic trees

were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/ TB2:S14355).

2.3. Molecular divergence estimates

5

Divergence time was estimated using a two-gene concatenated dataset with an

uncorrelated lognormal model incorporated in BEAST 1.6.1 (Drummond and Rambaut,

2007). No reliable ranid fossil record is presently available to provide proper calibration

within the genus Odorrana and therefore, 3 additional sequences of Rana bedriagae, R.

cretensis, and R. lessonae from Lymberakis et al. (2007) and 2 of R. signata and R.

chalconota from Bossuyt et al. (2006) were combined with our initial data and reanalyzed

with the aim to obtain a useful calibration. Divergence age estimates were established in this

study for R. cretensis and R. bedriagae (log-normal distribution with youngest of 5 MY and

standard deviation [SD] of 0.159) based on geological data (5-5.5MY) (Dermitzakis, 1990;

Beerli et al., 1996). Divergence between R. signata and R. chalconota (25MY, 1.264SD) was

estimated based on a multiple gene/calibration analysis (25-33MY) (Bossuyt et al. 2006;

Roelants et al. 2004). Analyses were executed with 20 million generations while sampling

every 1000th tree. Three identical BEAST runs were conducted to ensure the stability and

convergence of the MCMC chains. The results were combined using LogCombiner

(Drummond and Rambaut, 2007) and examined using Tracer 1.4 (Rambaut and Drummond,

2007) to evaluate stationarity. The first 10% of trees were discarded as burn-in. Molecular

rates for the two mtDNA genes were calculated using BEAST 1.6.1 (Drummond and

Rambaut, 2007) and compared with similar estimates from other vertebrates to cross-validate

our analysis of evolutionary dating.

3. Results and Discussion

3.1. Sequence characteristics

Sequence statistics and average nucleotide composition for the two gene fragments and

for the combined alignment are given in Table S2 in Appendix A. The total alignment for the

dataset included 1890 bp (12S = 789 bp; 16S = 1101 bp). Elimination of ambiguous sites

produced 754 bp for the 12S dataset and 1061 bp for the 16S dataset. A total of 805 out of

1815 sites were variable in the combined dataset, with 646 being parsimony informative

(35.6%). The average Ts/Tv ratio varied among genes and was 2.08 in the combined dataset

(Table S2 in Appendix A). Saturation analysis did not show any kind of saturation (data not

shown), and all substitutions of these two genes were therefore used for phylogenetic

6

reconstructions.

3.2. Phylogenetic relationships of Odorrana

The topologies of the ML and BI trees inferred from the analysis of the combined dataset

were identical, and both bootstrap support (BP) from ML and Bayesian posterior probability

(PP) are represented on the BI tree (Fig. 3). Our results support the sister-group relationship

of Babina and a clade containing all of the included Odorrana species, a finding consistent

with several recent studies (Che et al., 2007; Kurabayashi et al., 2010; Pyron and Wiens,

2011). Seven major branches within Odorrana were identified and denoted A-G (Fig. 3).

Odorrana chapaensis appears as the sister taxon to a clade comprising all other Odorrana

(CladeⅠ in Fig. 3), but cladeⅠ is not well supported (PP = 0.79, BP = 39). The result is thus

an unresolved three-way split between O. chapaensis and the two major subclades forming

Clade Ⅰ: Clade B (PP = 1.0, BP = 96) and Clade Ⅱ (PP = 0.99, BP = 75) in Fig. 3.

In Clade B, subclades B1 and B2 were recovered with strong supports (PP = 1.0, BP =

100). In subclade B1, the relationships among O. anlungensis, O. yizhangensis, and O.

lungshengensis could not be resolved with strong support. Clade B nonetheless rejects the

alliance of O. anlungensis, O. yizhangensis and O. lungshengensis with the O. schmacheri

group, a proposal based upon morphological data (Fei et al., 2009). Subclade B2 consisted of

the remaining species from southwestern China, the type species of the genus Odorrana, O.

margaretae and two species from Vietnam. Relationships within subclade B2 were

well-resolved except for one node (Fig. 3). The first phylogenetic split within subclade B2

separates Odorrana wuchuanensis from a clade comprising the remaining species (PP = 1.0,

BP = 100). Odorrana margaretae and O. kuangwuensis are grouped (PP = 0.99, BP = 80) as

the sister taxon to a clade comprising O. grahami, O. junlianensis, O. daorum, O.

hmongorum, O. andersonii and O. jingdongensis (PP = 1.0, BP = 100).

In Clade Ⅱ, O. ishikawae from Amami Island is the sister taxon to a highly supported

clade comprising the remaining species (PP = 1.0, BP = 98, Clade Ⅲ in Fig. 3). This position

of O. ishikawae among the Odorrana species was concordant with previous molecular

analyses (Matsui et al., 2005; Kurabayashi et al., 2010; Pyron and Wiens, 2011) but

contradicted with the results of Cai et al. (2007) as shown in Fig. 1C.

7

Clade Ⅲ consisted of nine species from southwestern to southeast China and O.

bacboensis (PP = 1.0, BP = 100, Clade D in Fig. 3) and a highly supported monophyletic

group (Clade Ⅳ in Fig. 3). The basal split within Clade D separated one well supported clade

O. tianmuii + O. huanggangensis (PP = 1.0, BP = 100, Fig. 3) from a more weakly supported

clade comprising O. bacboensis and the other seven species from southwestern to southeast

China (PP = 0.93, BP = 73, Fig. 3), within which interspecific relationships were well

resolved. In addition, O. bacboensis and O. tiannanensis are sister taxa (PP = 1.0, BP = 100),

and they have a close affinity with O. schmackeri, which contradicts prior result grouping O.

tiannanensis and O. livida (Cai et al., 2007; Pyron and Wiens, 2011) and the alliance of O.

tiannanensis with O. narina, O. tormota, O. nasica and O. versabilis (Kurabayashi et al.,

2010).

Clade E included O. chloronota, O. graminea, O. hosii, O. leporipes, O. banaorum and O.

morafkai (PP = 1.0, BP = 100), and interspecific relationships are well resolved. Clade E was

the sister clade to Clade Ⅴ (PP = 1.0, BP = 100) consisting of two well-supported

monophyletic groups (Clade F and G in Fig. 3). In agreement with the findings of recent

molecular analyses (Matsui, 2005; Stuart, 2008; Wiens et al., 2009; Pyron and Wiens, 2011),

data here clearly placed the O. livida complex and O. hosii well-nested within the genus

Odorrana.

Odorrana tormota, O. nasica, O. nasuta, O. exiliversabilis and O. versabilis formed a

monophyletic group (Clade F in Fig. 3), which was resolved as the sister group of Clade G

(PP = 1.0, BP = 100). In clade F, O. tormota, the concave-eared frog, is the sister taxon to a

highly supported clade comprising the remaining species (PP = 1.0, BP = 94). Odorrana

nasica is the sister species to O. exiliversabilis, O. versabilis and O. nasuta; among the latter

three species, the basal divergence separates O. exiliversabilis from O. versabilis and O.

nasuta (PP = 1.0, BP = 99). The inclusion of O. tormota and O. nasica in the genus Odorrana

was largely congruent with several molecular studies (Cai et al., 2007; Stuart, 2008; Wiens et

al., 2009; Kurabayashi et al., 2010; Pyron and Wiens, 2011).

Clade G contained O. amamiensis, O. supranarina, O. narina, O. utsunomiyaorum and O.

swinhoana from two different localities in Taiwan, China (PP = 1.0, BP = 96). Odorrana

amamiensis, O. supranarina, and O. narina formed a monophyletic group (PP = 1.0, BP =

8

100), within which O. amamiensis and O. narina were grouped as sister species (PP = 1.0,

BP = 99), a finding concordant with several previous molecular studies (Matsui et al., 2005;

Pyron and Wiens, 2011).

3.3. Divergence-time estimation

According to our estimates, we obtained an average rate for the two mtDNA genes of

0.746% per lineage per million years for all substitutions, and this rate is comparable to those

(0.5-1%/million years) in other vertebrates (Caccone et al., 1997). The divergence dates

inferred by the Bayesian relaxed clock analyses suggest that the genus Odorrana began to

diversify 18.99 million years ago (95% HPD interval 14.90-23.23 MYA), during the Late

Oligocene to Middle Miocene (Fig. S1 and Table S3 in Appendix A), which conflicted

dramatically with the Late Ecocene split approximately 38 MYA proposed by Wiens et al.

(2009) based on the ranid crown-group calibration. The earliest split within Odorrana,

between O. chapaensis and the remaining Odorrana species, is estimated at 14.44 MYA

(11.26-18.01 MYA) during the Early to Middle Miocene (Fig. S1 and Table S3 in Appendix

A). The divergence time between O. ishikawae and its sister clade is estimated to be 13.22

MYA (10.21-16.40 MYA), which accorded closely with the divergence at 12.6 (7.9-18.0)

MYA predicted by Matsui et al. (2005). In addition, the origination of the other major clades

(Clades B, D to G in Fig. 3) within Odorrana occurred in the Early to Late Miocene, and

each of the major clades underwent a radiation from the Middle Miocene to the Pleistocene,

with most divergences in the Plio-Pleistocene (Fig. S1 and Table S3 in Appendix A). The

divergence times predicted in the present study were slightly older or younger than other

studies in some lineages, but overall these findings were consistent with or overlapped in

range the previous estimates (Matsui et al., 2005; Wiens et al., 2009). Climate change from

greenhouse to icehouse, plate tectonic movements, and the uplift of mountain ranges might

have played a key role in the Odorrana radiation (Matsui et al., 2005; Molnar, 2005;

Metcalfe, 2006; Hall, 2009).

Acknowledgments

Financial support was provided by the National Natural Science Foundation of China

9

(NSFC) to XHC (grant no. 30870277), GMZ (grant no. 30170187), and KYZ (grant no.

30470249), as well as by the Scientific Research Foundation of HNNU to ZC (grant no.

01046500138). Permission for field surveys were granted by the Administrative Office of

Wuyi Mountains National Nature Reserve of Fujian Province, Jiangxi Wuyishan National

Nature Reserve Administrative Office, Hainan Jianfengling Forestry Park. We are grateful to

J. Yang, L. Li, J. Tao, F.X. Zeng, Z.L. Jiang, L.K. Tao and C.G. Zhu for their help in field

trips. We thank H.T. Shi, L.J. Wang, W.H. Chou, J.C. Wang, Y.C. Zheng, D.Q. Rao and B.R.

Geng for their assistance in sample collection. We thank J.Z. Fu, J. Yan, J.J. Li, C. Tian, J.X.

Xu, W.H. Yu, and X.M. Zhou for their technical assistance. We also thank Dr. A. Larson, and

two anonymous reviewers for insightful comments on this manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at doi:

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Figure captions

Fig. 1 Alternative hypotheses of phylogenetic relationships within the genus Odorrana as

obtained from morphological and molecular sequence data.

Fig. 2 Map of East and Southeast Asia showing sampling localities of Odorrana included in

this study. Locality numbers are presented in Table 1.

Fig. 3 Phylogenetic trees reconstructed using BI and ML methods based on the concatenated

dataset of 12S and 16S rRNA for species of Odorrana and related species. Integers associated

with branches are bootstrap support values for ML inference whereas values of 1 or less are

Bayesian posterior probabilities. Representative members are delimited by vertical lines to

the right of the tree. Numbers in parentheses correspond to those of localities in Table 1 and

Fig. 2. The topotypes analyzed in this study are shown by bold, and asterisk after taxon name

indicates paratypes.

Fig. S1 Time-calibrated Odorrana phylogeny derived from BEAST using the concatenated

dataset of 12S and 16S rRNA for species of Odorrana and related species. Clade letters are

identical to those in Table S3. Red boxes indicate nodes for which a prior calibration

constraint distribution was used, and blue boxes indicate divergence dates estimated without

prior calibration constraints for that node. The bounds of the boxes correspond to the 95%

highest posterior density (HPD) of each node. Numbers in parentheses correspond to those of

localities in Table 1 and Fig. 2.

14

Table 1. Samples and sequences used in this study.

Family Genus Species Locality Voucher GenBank Accession numbers

(Frost, 2013) (Fei et al., 2009; Frost, 2013) 12S rRNA gene 16S rRNA gene

Ingroup

Ranidae Odorrana 1 Odorrana exiliversabilis Wuyishan, Fujian HNNU0607032 topotype KF185020 KF185056

2 Odorrana nasuta Wuzhishan, Hainan HNNU051119 topotype KF185017 KF185053

3 Odorrana versabilis Leishan, Guizhou HNNU003 LS topotype KF185019 KF185055

4 Odorrana swinhoana Taibei, Taiwan HNNUTW1 KF185009 KF185045

5 Odorrana swinhoana Nantou, Taiwan HNNUTW9 KF185010 KF185046

6 Odorrana leporipes Shaoguan, Guangdong HNNU1008Ⅰ099 topotype KF185000 KF185036

7 Odorrana graminea Wuyishan, Fujian HNNU0607051 KF185003 KF185039

8 Odorrana graminea Wuzhishan, Hainan HNNU0606123 topotype KF185002 KF185038

9 Odorrana graminea Junlian, Sichuan HNNU014JL KF185001 KF185037

10 Odorrana hainanensis Wuzhishan, Hainan HNNU0606105 topotype KF184996 KF185032

11 Odorrana sp.1 Shiwanshan, Guangxi HNNU 2957k KF184997 KF185033

12 Odorrana tiannanensis Hekou, Yunnan HNNUHK001 topotype KF185008 KF185044

13 Odorrana schmackeri Yichang, Hubei HNNU 0908Ⅱ349 topotype KF185011 KF185047

14 Odorrana nanjiangensis Nanjiang, Sichuan HNNU1007Ⅰ291 topotype KF185006 KF185042

15 Odorrana hejiangensis Hejiang, Sichuan HNNU1007Ⅰ202 topotype KF185016 KF185052

16 Odorrana sp.2 Yichang, Hubei HNNU1007Ⅰ061 KF185005 KF185041

17 Odorrana tianmuii Linan, Zhejiang HNNU 0707071 paratype KF185004 KF185040

18 Odorrana huanggangensis Wuyishan, Fujian HNNU0607001 paratype KF185023 KF185059

19 Odorrana grahami Kunming, Yunnan HNNU1008Ⅱ016 topotype KF185015 KF185051

20 Odorrana junlianensis Junlian, Sichuan HNNU002 JL topotype KF185022 KF185058

21 Odorrana andersonii Longchuan, Yunnan HNNU001YN topotype KF185021 KF185057

22 Odorrana jingdongensis Jingdong, Yunan 20070711017 topotype KF185014 KF185050

23 Odorrana kuangwuensis Nanjiang, Sichuan HNNU 0908Ⅱ185 topotype KF184998 KF185034

24 Odorrana margaretae Emei, Sichuan HNNU20050032 KF184999 KF185035

25 Odorrana wuchuanensis Wuchuan, Guizhou HNNU019 L topotype KF185007 KF185043

26 Odorrana yizhangensis Yizhang, Hunan HNNU1008Ⅰ075 topotype KF185012 KF185048

27 Odorrana lungshengensis Longsheng, Guangxi HNNU70028 topotype KF185018 KF185054

28 Odorrana anlungensis Anlong, Guizhou HNNU1008Ⅰ109 topotype KF185013 KF185049 29 Odorrana chapaensis Lai Chau, Vietnam Genbank DQ283372 DQ283372

15

30 Odorrana chloronota Ha Giang, Vietnam Genbank DQ283394 DQ283394 31 Odorrana nasica Ha Tinh, Vietnam Genbank DQ283345 DQ283345 32 Odorrana tormota Huangshan, Anhui Genbank, topotype DQ835616 DQ835616 33 Odorrana hosii Kuala Lumpur, Malaysia Genbank AB511284 AB511284 34 Odorrana narina Okinawa Island, Japan Genbank AB511287 AB511287 35 Odorrana ishikawae Amami Island, Japan Genbank AB511282 AB511282 36 Odorrana daorum Sa Pa, Vietnam Genbank AF206101 AF206482 37 Odorrana bacboensis Khe Moi, Nghe An, Vietnam Genbank AF206099 DQ650569 38 Odorrana hmongorum Lao Cai, Vietnam Genbank -- EU861559 39 Odorrana amamiensis Tokunoshima, Ryukyu Genbank AB200923 AB200947 40 Odorrana banaorum Tram Lap, Vietnam Genbank AF206106 AF206487 41 Odorrana morafkai Tram Lap, Vietnam Genbank AF206103 AF206484 42 Odorrana supranarina Iriomotejima, Ryukyu Genbank AB200926 AB200950 43 Odorrana utsunomiyaorum Iriomotejima, Ryukyu Genbank AB200928 AB200952 Outgroup

Ranidae Babina Babina adenopleura Wuyishan, Fujian HNNU0607055 KF185028 KF185064

Babina daunchina Emeishan, Sichuan HNNU20060103 topotype KF185029 KF185065

Babina lini Lvchun, Yunnan HNNULC001 KF185030 KF185066

Babina holsti Okinawa Island, Japan Genbank AB511296 AB511296

Hylarana Hylarana guentheri Fuzhou, Fujian HNNU060435 KF185024 KF185060

Hylarana spinulosa Wuzhishan, Hainan HNNU051117 KF185031 KF185067

Pelophylax Pelophylax nigromaculata Jinzhai, Anhui HNNU F97062 KF185026 KF185062

Pelophylax plancyi Genbank NC_009264 NC_009264

Rana Rana chensinensis Ningshan, Shanxi HNNU 20060359 KF185025 KF185061

Rugosa Rugosa tientaiensis Tianmushan, Zhejiang HNNU 98606 KF185027 KF185063

a) b)

c) d)

e) f)

g) h)

Ye and Fei, 2001 Matsui et al., 2005

Cai et al., 2007 Che et al., 2007

Stuart, 2008 Wiens et al., 2009

Kuarabayashi et al., 2010 Pyron and Wiens, 2011

Odorrana hainanensisOdorrana margaretaeOdorrana andersoniiOdorrana grahamiOdorrana kuangwuensisOdorrana wuchuanensisOdorrana lividaOdorrana exiliversabilis

Odorrana versabilisOdorrana nasuta

Odorrana hejiangensisOdorrana anlungensisOdorrana tiannanensisOdorrana schmackeri

Odorrana swinhoanaOdorrana lungshengensis

Odorrana margaretaeOdorrana grahamiOdorrana ishikawaeOdorrana hosii

Odorrana lividaOdorrana chloronotaOdorrana hejiangensis

Odorrana schmackeri

Odorrana swinhoanaOdorrana utsunomiyaorum

Odorrana supranarinaOdorrana amamiensisOdorrana narina

Odorrana ishikawaeOdorrana chapaensisOdorrana margaretaeOdorrana grahamiOdorrana andersoniiOdorrana daorumOdorrana hmongorumOdorrana schmackeriOdorrana hejiangensisOdorrana tiannanensisOdorrana morafkaiOdorrana banaorumOdorrana hosiiOdorrana lividaOdorrana chloronotaOdorrana tormotaOdorrana nasicaOdorrana versabilisOdorrana utsunomiyaorumOdorrana swinhoanaOdorrana supranarinaOdorrana amamiensisOdorrana narina

Odorrana chapaensis

Odorrana margaretae

Odorrana andersonii

Odorrana grahami

Odorrana hejiangensis

Odorrana schmackeri

Odorrana livida (China)

Odorrana nasica

Odorrana versabilis

Odorrana livida (Vietnam)

Odorrana chapaensis

Odorrana bacboensis

Odorrana grahami

Odorrana hmongorum

Odorrana margaretae

Odorrana tormota

Odorrana nasica

Odorrana hosii

Odorrana morafkai

Odorrana chloronota

Odorrana livida

Odorrana chapaensisOdorrana margaretaeOdorrana grahamiOdorrana andersoniiOdorrana hejiangensisOdorrana schmackeriOdorrana ishikawaeOdorrana bacboensisOdorrana chloronotaOdorrana lividaOdorrana hosiiOdorrana banaorumOdorrana morafkaiOdorrana tormotaOdorrana versabilisOdorrana nasicaOdorrana swinhoanaOdorrana utsunomiyaorumOdorrana supranarinaOdorrana amamiensisOdorrana narina

Odorrana chapaensisOdorrana andersonii

Odorrana margaretaeOdorrana grahami

Odorrana ishikawaeOdorrana schmackeriOdorrana hejiangensisOdorrana hosiiOdorrana lividaOdorrana tiannanensisOdorrana narinaOdorrana tormotaOdorrana nasicaOdorrana versabilis

Odorrana chapaensisOdorrana daorumOdorrana jingdongensisOdorrana hmongorumOdorrana andersoniiOdorrana junlianensisOdorrana grahamiOdorrana margaretaeOdorrana ishikawaeOdorrana hejiangensisOdorrana schmackeriOdorrana bacboensis

Odorrana tiannanensisOdorrana banaorumOdorrana morafkaiOdorrana lividaOdorrana chloronotaOdorrana hosiiOdorrana tormotaOdorrana nasicaOdorrana versabilisOdorrana swinhoanaOdorrana utsunomiyaorumOdorrana supranarinaOdorrana amamiensisOdorrana narina

Figure 1

Figure 1

Figure 2

Odorrana swinhoana (4)Odorrana swinhoana (5)

Odorrana utsunomiyaorum (43)Odorrana narina (34)

Odorrana amamiensis (39)Odorrana supranarina (42)

Odorrana nasuta (2)Odorrana versabilis (3)

Odorrana exiliversabilis (1)Odorrana nasica (31)

Odorrana tormota (32)Odorrana leporipes (6)

Odorrana graminea (7)Odorrana graminea (8)

Odorrana graminea (9)Odorrana chloronota (30)

Odorrana hosii (33)Odorrana banaorum (40)Odorrana morafkai (41)

Odorrana hainanensis (10)Odorrana sp. 1 (11)

Odorrana tiannanensis (12)Odorrana bacboensis (37)

Odorrana schmackeri (13)Odorrana nanjiangensis (14)Odorrana hejiangensis (15)

Odorrana sp. 2 (16)Odorrana tianmuii (17)

Odorrana huanggangensis (18)Odorrana ishikawae (35)

Odorrana grahami (19)Odorrana junlianensis (20)

Odorrana daorum (36)Odorrana hmongorum (38)

Odorrana andersonii (21)Odorrana jingdongensis (22)

Odorrana kuangwuensis (23)Odorrana margaretae (24)

Odorrana wuchuanensis (25)Odorrana yizhangensis (26)Odorrana lungshengensis (27)

Odorrana anlungensis (28)Odorrana chapaensis (29)

Babina adenopleuraBabina lini

Babina daunchinaBabina holsti

Rana chensinensisPelophylax nigromaculataPelophylax plancyiHylarana spinulosaHylarana guentheri

Rugosa tientaiensis

57/0.56100/1.00

96/1.00

100/1.00

99/1.00

100/1.00

94/1.00

99/1.0094/1.00

99/1.00

100/1.00

100/1.00100/1.00

99/1.00

99/1.0096/1.00

100/1.00

98/1.00

100/1.00

100/1.00100/1.00

100/1.00

100/1.00

100/1.00100/1.00

73/0.93

77/0.96

75/0.99

39/0.79

96/1.00

100/1.00

55/0.62

100/1.00

100/1.00

80/0.99

63/0.8477/0.94 97/1.00

77/0.9682/1.00

90/0.99

99/1.00100/1.00

57/0.83

100/1.00

68/0.84

76/0.95

0.04

32/0.55

ana r

rod

O

Babina

RanaPelophylaxHylaranaRugosa

100/1.00

A

B

C

D

E

F

G

* * eadi

naR

B1

B2

Ⅱ

Ⅲ

Ⅳ

Ⅴ

Ⅰ

Figure 3

0.04

Odorrana

Ranidae

Babina

RanaPelophylaxHylaranaRugosa

A

B

C

D

E

F

G

*Graphical Abstract

Highlights

1. Molecular phylogeny of the genus Odorrana with many paratypes and topotypes

2. The basal position of Odorrana chapaensis among the Odorrana species

3. Phylogenetic evidence for the monophyly of seven major branches of Odorrana

4. Interrelationships within Odorrana and rapid divergence of Odorrana.